FMRFamide receptors in Helix aspersa

Peptides, Vol. 8, pp. 1065--1074. ©PergamonJournals Ltd., 1987. Printed in the U.S.A.

0196-9781/87 $3.00 + ,00

FMRFamide Receptors in Helix aspersa KEMAL

PAYZA

The Whitney Laboratory, University o f Florida, 9505 A I A South, St. Augustine, F L 32086

R e c e i v e d 7 J u l y 1987 PAYZA, K. FMRFamide receptors in Helix aspersa. PEPTIDES 8(6) 1065-1074, 1987.--An in vitro receptor binding assay and an isolated heart bioassay were developed and used to characterize the structure-activity relations (SAR) of FMRFamide receptors in a land snail, Helix aspersa. In the radioreceptor assay, binding of 12'~I-desaminoTyr-PhenorLeu-Arg-Phe-amide (~2~I-daYFnLRFamide) at 0°C to Helix brain membranes was reversible, saturable, and specific, with a Ko of 14 nM and a Bm,~xof 85 fmol/mg brain. A lower affinity site was also observed (KD= 245 nM; Bmax=575 fmol/mg brain). In the heart bioassay, daYFnLRFamide and other FMRFamide analogs increased myocardial contraction force, The SAR of cardiostimulation correlated with the specificity of high affinity '2'~I-daYFnLRFamide binding to brain and heart receptors. The SAR was also similar to that described for other molluscan FMRFamide bioassays, except for a marked preference for N-blocked analogs. Peptides with N-terminal extensions of desaminoTyr, Tyr, Tyr-Gly-Gly, and acetyl, exhibited the highest potency in both radioligand displacement and cardiostimulation. The endogenous Helix heptapeptide analogs of FLRFamide (pQDP-, NDP-, and SDP-FLRFamide) were stimulatory on the heart at low doses, but were inhibitory at moderate to high doses. These peptides were 20 times weaker than FMRFamide in both the brain and heart receptor binding assays, with IC.~osabout 10 p.M. The results suggest that the effects of FMRFamide in Helix are receptor-mediated, and that the heptapeptides do not interact at FMRFamide receptors, FMRFamide receptors

Radioreceptor assay

Helix neurons

THE FMRFamide-related peptides (FaRPs) of the pulmonate gastropod mollusc Helix aspersa are: the tetrapeptides FMRFamide [17] and F L R F a m i d e [19], and the heptapeptides p Q D P F L R F a m i d e [17], N D P F L R F a m i d e [18], and S D P F L R F a m i d e [19]. The pharmacological effects of the heptapeptides on the neurons [1,3], heart [5,17], and retractor muscles [II] of Helix are distinct from those of FMRFamide. These results suggest that FMRFamide receptors and heptapeptide receptors occur and function separately in Helix tissues. The aim of the present study was to identify and characterize the putative FMRFamide receptors in the heart and brain of Helix. The methods involved developing a bioassay of the isolated heart and a radioligand binding assay for FMRFamide receptors in brain membranes. The ligand, an analog of F M R F a m i d e (daYFnLRFamide), was synthesized by coupling the Bolton-Hunter reagent (an active ester of desaminotyrosine) to the N-terminal of FnLRFamide. The resulting peptide could then be iodinated on the desaminoTyr without oxidative damage. The ligand also lacks the N-terminal amino group required by aminopeptidases which, in clam heart membranes, have been shown to degrade FMRFamide rapidly [10]. The structure-activity relations (SAR) of Helix brain and heart F M R F a m i d e receptors were determined by measuring the potencies of FMRFamide analogs in both (a) displacing '25I-daYFnLRFamide bound to Helix brain and heart membranes, and (b) stimulating the isolated, perfused heart. The potencies of peptides in displacing bound radioligand were correlated with those for stimulating the isolated heart. Preliminary reports of these experiments have been presented recently [14,15].

Molluscan heart

METHOD Live Helix aspersa were purchased from Dr. Robert Koch (Fullerton, CA), and from Night Bird Game and Fowl Co. (San Francisco, CA). Peptides were purchased from Sigma, Peninsula, Bachem, and CRB, except for FMRWamide, FMRLamide, and YGGFMRamide, which were synthesized by Dr. J. P. Riehm (University of West Florida, Pensacola, FL). The following materials were from Sigma: bovine serum albumin (BSA); chloramine T; triethylamine (TEA); dimethylformamide (DMF); 3iodo-L-tyrosine; Bolton-Hunter reagent; and the buffers piperazine-N,N'-bis[2-hydroxypropane-sulfonic acid] (POPSO), N-[2-hydroxyethyl]-piperazine-N'-3-propane-sulfonic acid (EPPS), N,N-bis[2-hydroxyethyl]-glycine (BICINE), N-tris [hydroxymethyl]methyl-glycine (TRICINE), 3-[N-tris0aydroxymethyl)-methylamino]-2-hydroxypropane-sulfonic acid (TAPSO), and tris(hydroxymethyl)aminomethane (TRIS). Diiodo-L-tyrosine was from Nutritional Biochemicals. NanSI was obtained from ICN. The reagents TEA and D M F were distilled prior to use. Fast atom bombardment/mass spectroscopy(FAB/MS) of I-daYFnLRFamide was performed by Dr. Terry Lee (Beckman Research Institute of the City of Hope, Duarte, CA). Helix Heart Bioassay

Helix hearts were dissected out and mounted in the bioassay apparatus (Fig. 1) with a cannula inserted through the auricle and just into the ventricle. Each heart was perfused at 200 /zl/min with saline (75 mM NaC1, 4 mM KC1, 7 mM CaClz, 5 mM MgCI2, 5 mM glucose, 0.1% BSA, 5 mM Tris, pH 7.5) which flowed out the aorta to bathe the surface of the

1065

1~6

PAYZA

IFT

0.02

SRI b-

17.61

< 2_2.61

HH

0.01

L_

SH

SA

~" 30 0 0

20

E m

9

WA FIG. 1. Apparatus for isolated perfused Helix heart bioassay. The Helix heart (HH) is mounted on a cannula inserted into the ventricle, and is perfused under constant pressure from a saline reservoir (SR). The aorta is threaded to a force transducer (FT). Valves are shown directing the flow of saline through the shunt loop (SH) during loading of a peptide solution into the sample loop (SA), with the excess flowing to waste (WA). The sample is carried to the heart, without interrupting the flow, upon readjustment of the valves. heart. The aorta was snared by a thin glass hook which was threaded to a force transducer; the output was recorded with a Grass Model 79C polygraph. The Helix heart preparations were spontaneously active, and could remain beating for many hours. Aliquots of peptides at known concentrations were introduced to the perfusing stream without perturbing its flow. This was done by first diverting the perfusate through the shunt loop (Fig. 1.), then injecting the peptide solution into the 400/xl sample loop, and finally switching the valves back so that the sample would be carried to the heart. The response to a dose of peptide was measured as the increase in myocardial contractile force above the control amplitude. Each response was expressed as a percentage of the maximal effect (Emax). From log dose-response curves, the concentration producing half-maximal effect (ECso) was obtained, and the potency of each peptide was expressed in terms of this parameter. Preparation of 125I-daYFnLRFarnide The desaminotyrosinyl derivative of F n L R F a m i d e was prepared and purified as follows. One/.tmole peptide and 1

o I0

Fraction

20

30

no. (I min)

FIG. 2. HPLC analysis of daYFnLRFa, I-daYFnLRFa, and 125IdaYFnLRFa. Peptides were synthesized, purified by reverse-phase TFA/ACN HPLC as described in the Method section, and rechromatographed to illustrate separation of daYFnLRFa (17.61 min) and I-daYFnLRFa (22.61 min). Lower chromatogram shows radioactivity collected per 1 minute fraction, and demonstrates coelution of purified '25I-daYFnLRFa with the peak identified as the mono-iodo derivative. /zmole T E A were allowed to react with 3 /zmoles BoltonHunter reagent for 4 hr at 4°C in 100/xl DMF. The mixture was then chromatographed in 5 mM phosphate buffer (K ÷ salt, pH 7) at 2 ml/min through a Cls reverse-phase HPLC column (Waters Novapak), with acetonitrile (ACN) as the organic solvent (initial: 6% ACN; 5 min: 30% ACN; 20 min: 48% ACN). The d a Y F n L R F a m i d e product was identified and distinguished from the reactants and other side-products by: (a) its elution at about 13 minutes, compared to 8 minutes for F n L R F a m i d e ; (b) its absorbance at 278 nm due to the added desaminotyrosinyl group; (c) its immunoreactivity with rabbit anti-YGGFMRFamide antiserum [16]; (d) its biological activity on isolated Mercenaria and Helix hearts; and (e) its capacity for iodination. The product peak was rechromatographed at pH 4 in 0.075% trifiuoroacetic acid (TFA), with another sequential gradient (initial: 8% ACN; 5 rain: 20% ACN; 20 min: 40% ACN; 25 min: 72% ACN). The purified d a Y F n L R F a m i d e , which eluted at about 18 minutes with this T F A / A C N system (Fig. 2), was dried and redissolved in water. The concentration was quantitated by subject-

FMRFAMIDE RECEPTORS IN HELIX ASPERSA

1067 FnLRFamide generated upon loss of the iodine from the peptide.

E "o i¢) O

Preparation of Brain Membranes

Z Z 133

t\

It. O laJ (3_ 03

~ A

-r

0

i

1

I

~

A !

T

i

2

~

!

3

TIME, hours FIG. 3. Effect of temperature on 12~I-daYFnLRFamidebinding to Helix brain membranes. Membranes were incubated with 0.5 nM X2~l-daYFnLRFamide in the absence or presence of 100 ~M acFnLRFamide at 0°C (0) and 25°C (A). At the times indicated, the specific binding was determined as described in the Method section, and has been plotted against incubation time. ing aliquots to TFA/ACN HPLC, and comparing the Azr8 of the peptide to a standard curve of YGGFMRFamide. The iodination of daYFnLRFamide proceeded as follows [7]. To a polypropylene microcentrifuge vial containing daYFnLRFamide (1-2.5 nmoles, depending on the desired specific activity) in 10/zl of 0.5 M K/PO4 buffer (pH 7), one nmole of Na125I in 3-4 p.1 0.1 N NaOH was added, followed by 5/zl chloramine T (2 mg/ml in phosphate buffer). After being mixed for one minute, the reaction was terminated with I00/zl sodium metabisulfite (5 mg/ml buffer), and the mixture was applied to a prepared Cas Sep-pak cartridge (Waters). The free iodide was washed through with 20 ml water. The unreacted and iodinated peptides were eluted together with 5 m180% ACN, and collected as 1 ml fractions. The radioligand (800--2000 Ci/mmole) was stored at -5°C. To purify the mono- and di-iodinated analogs, an aliquot of the trace was subjected to reverse-phase TFA/ACN HPLC as above, and the peaks of radioactivity corresponding to ~2~I-daYFnLRFamide (23 min elution time) and [125I]2-daYFnLRFamide (26 min) were collected. This assignment of peaks was based on: (a) the increases in retention time associated with substitution of iodine into the peptide; and (b) coelution of 'z~I-daYFnLRFamide with its purified nonradioactive homolog (Fig. 2). The nonradioactive I-daYFnLRFamide, used in homologous displacement (i.e., saturation) experiments, was produced with an upscaled version of the above iodination method. The mono-iodo peptide was separated from the unreacted and di-iodo analogs by TFA/ACN HPLC; the peaks were first identified by comparing their uv absorption spectra to those of standard 3-iodo-L-tyrosine and 3,5 diiodo-L-tyrosine. The uniodinated peptide has a peak of absorption at 275 nm, whereas I-daYFnLRFamide showed a maximum at 282 nm; this peak corresponded to that of monoiodo-tyrosine (281 nm). The peak for the diiodo derivatives was shifted to 285 nm. Finally, FAB/MS of the monoiodo peptide showed a molecular weight ion of 855, thus confirming the composition as I-daYFnLRFamide. A scond ion, of molecular weight 729, corresponded to daY-

Brains (circumesophogeal ganglia) from live Helix were dissected out, washed in saline, weighed, and homogenized with a Polytron homogenizer for 15 seconds in 50 ml hypotonic buffer/g tissue (I0 mM POPSO, pH adjusted to 7.9 with KOH). This suspension was spun at 30,000×g for 10 minutes at 4°C, and the pellet was resuspended in 200 mM EPPS pH 7.9. Following incubation at 25°C for 20 minutes, the suspension was spun as before, and the pellet was washed and spun twice in 50 mM POPSO (this procedure maximized the potency of acFnLRFamide in the binding assay). The final pellet was then resuspended in assay buffer (80 mM POPSO, 1% BSA, pH 7.9, 30 ml/g tissue).

~251-daYFnLRFamide Binding Assay Incubations (in triplicate) proceeded in 300/~1 assay buffer (described above) at 0°C in 12×75 mm test tubes. Aliquots (150/zl) of membrane suspension were added to a mixutre of 100/xl ~5I-daYFnLRFamide and 50/xl containing either: assay buffer for determining total binding (TB); or acFnLRFamide (100/~M final concentration) for measuring nonspecific binding (NS); or other peptides at various concentrations in assay buffer, for the competitive displacement studies. After 2.5 hr at 0°C, the contents of each tube were filtered and washed rapidly with 3 ×4 ml ice-cold wash buffer (80 mM Tris, 1% BSA, pH 7.3) through pre-soaked Whatman GF/B filters mounted in Millipore vacuum manifolds. The radioactivity (dpm) bound to the membranes retained on the filters was measured in a Beckman 7000 gamma counter. Specific binding, the difference between total and nonspecific binding, was expressed as either dpm/filter or fmol/mg brain, and represented about 70% of the total binding. The amount of tissue per tube was such that less than 10% of the total radioligand was bound at equilibrium. Saturation data were plotted by the method of Scatchard [20] and analyzed with a nonlinear curve-fitting computer program originally developed for pharmacokinetic modelling [21]. The program fit the saturation data iteratively as a summation of two independent binding sites (r=0.99). The potency of each peptide tested was expressed as the IC~o(the concentration that inhibits 50% of the specific binding), which was obtained from plots of % SB vs. Log [Peptide].

Optimization of Binding Assay The assay procedure described above was based on the following observations: (1) At 25°C, incubation of ~25I-daYFnLRFamide with Helix brain membranes resulted first in an increase and then a decrease in specific binding (Fig. 3). When the radioligand was extracted from the 25°C incubation mixture and tested in the RIA [16], a loss of binding with the antiYGGFMRFamide antiserum was observed. Further analysis of the extract by HPLC revealed a drop in the peak of 1251daYFnLRFamide and the appearance of three fragments, coeluting with ~25I-daYF, 125I-daYFnL, and 125I-daYFnLR (standards were generated with peptidases obtained from Sigma). However, the degradative activity of the membranes could be inhibitec l by reducing the temperature to 0°C; after three hours at 0°C, 'mor~ than 95% of the trace (or other peptides used in the competi,t,iori studies) remained intact, as assayed by HPLC. Therefore, the binding assay was con-

1068

PAYZA

I00

-

0

/

x 0

E LLI

5O

/

A 1 FMRFomide 2xlO-6M

YFMRFomide 2xlO-7M

doYFMRFomide 2xlO-SM

B

VY

1 FMRF 2xlO-6M i

-8

i

-7

i

-6

i

-5

Log Molor Peptide Concentrotion FIG. 4. Effect of N-acetylation on stimulation of isolated Helix heart. Responses of the heart to FnLRFamide (O) and acFnLRFamide (A) were measured, and %E,,,x has been plotted against log of the peptide concentration in the perfusate. Acetylation of FnLRFamide increased potency from 1.3 to 0.042 #M. ducted at 0°C, resulting in a time-dependent saturation of specific binding (Fig. 3). The preparation of the brain membranes included a preincubation at 25°C to effect the degradation of any endogenous peptides; the 20 minute period was sufficient for the membranes to degrade 90% of I nmole 'zsI-YGGFMRFamide added as test substrate. (2) Specific binding of the mono-iodinated radioligand was about 10 times that of the di-iodo ligand, and its nonspecific binding was 35% lower. The proportion of the trace consisting o f the mono-iodo peptide was maximized, therefore, either by iodinating to a lower specific activity (i.e., decreasing the molar ratio of iodide to peptide from 1 to 0.4), or by isolating the mono-iodo peptide through reverse phase HPLC (Fig. 2). (3) BSA was required in both the assay and wash buffers in order to reduce the nonspecific binding; a concentration of I% (w/v) was maximally effective. (4) The buffer POPSO (80 mM, pH 7.9) was optimal for the binding incubation at 0°C. The buffers tested and their relative effectivenesss were: POPSO > EPPS > BICINE = TRICINE > TAPSO = TRIS. Nevertheless, TRIS could be substituted for POPSO in the wash procedure with no loss in binding, but a substantial saving in cost. For the preincubation of the membranes at 25°C, 200 mM EPPS resulted in maximal specific binding. (5) Preliminary experiments showed that filtration was superior to centrifugation for separating free radioligand from that bound to the brain membranes, because filtration resulted in lower nonspecific binding. (6) The specific binding (SB) and the ratio SB/TB were maximal after 3 or 4 washes of 4 ml wash buffer. Three washes were used to conserve materials. RESULTS

SAR o f Helix Heart Bioassay

The Helix heart responded to FMRFamide analogs with a dose-dependent increase in contractile force (Fig. 4). The response was also dependent on peptide composition. For

NDPFLRFomide 10-8M

MRFomide 10-5M

FIG. 5. Responses of isolated Helix heart to FMRFamide analogs. Peptides were introduced to the saline perfusing an isolated Helix heart (Fig. 1) for 2 min, and relative contraction force (vertical direction) was measured as described in the Method section. Recording (A) illustrates the enhancement of potency with N-terminal extensions of Tyr and desaminoTyr. Responses unlike that of FMRFamide are shown in (B). example, the C-terminal amide was required for stimulation; i.e., the free acid F M R F - O H was inactive at concentrations up to 10 tzM. Similarly, there was a requirement for the N-terminal Phe; the fragment MRFamide lacked activity up to 10/zM, and produced a response qualitatively unlike that of FMRFamide at or above 10 p.M (see Fig. 5). The SAR of FMRFamide-induced cardiostimulation is presented in Table 1 (EC~0s) and is discussed below. The effects of amino acid substitutions at the first two positions, and of N-terminal extensions, were examined. At the Phe' position, tryptophan substitution increased potency by twofold, without affecting E . . . . The norleucine 2 analog (FnLRFamide) was equipotent with FMRFamide, but replacing the Met z with leucine decreased potency twofold. Similarly, a group of daY derivatives with Met substitutions showed the same rank order of potency (Met = norLeu > Leu). Certain N-terminal extensions enhanced potency without affecting Em~,x, in the order: desaminoTyr > acetyl > TyrGly-Gly > Tyr (Table 1, Figs. 4 and 5A). The greatest effect, obtained with the desaminoTyr extension, was an increase in potency by about 100-fold. The heptapeptide Y G G F M R F amide (met-enkephalin-Argn-PheT-amide), which has been reported to occur in molluscs [12,22], was about 10× as potent as FMRFamide. In contrast to most N-terminally extended analogs, the Helix heptapeptides p Q D P F L R F a m i d e , N D P F L R F a m i d e , and S D P F L R F a m i d e produced biphasic responses on the heart. These peptides were 30 to 50x more bioactive than FMRFamide at low doses, but were strongly inhibitory above 0.01 to 0.03/zM (Fig. 5B). 1251-daYFnLRFamide Binding to Helix Brain Membranes

The binding of '25I-daYFnLRFamide to Helix brain membranes at 0°C was reversible (Fig. 6) and proportional to tissue concentration (Fig. 7). Saturation experiments with increasing concentrations of I-daYFnLRFamide and uniodinated d a Y F n L R F a m i d e were performed; Scatchard plots [20] of the binding data were curvilinear (Fig. 8). Fits to a two-site model [21] yielded a relatively high affinity site

FMRFAMIDE RECEPTORS IN HELIX ASPERSA

1069

TABLE 1 POTENCIES OF PEPTIDES FOR Helix FMRFamide RECEPTORS IC~,, (/~M)~: Structural Change --

Peptide Composition*

EC.~ot (/xM)

Brain

FMRFa

1.3

0.53

Phe 1 substitution

WnLRFa YMRFa

0.60

0.60 1.4

Met 2 substitution

FnLRFa FLRFa F(M-Ox)RFa

Arg 3 substitution

FMKFa

2.0

Phe 4 substitution

FMRWa FMRLa

0.20 4.0

N-terminal deletions

N-terminal extentions

MRFa RFa acFa Fa

1.3 2.6

§

daYFMRFa daYFnLRFa I-daYFnLRFa daYFLRFa YFMRFa acFnLRFa

0.010 0.011

Helix heptapeptides

NDPFLRFa pQDPFLRFa SDPFLRFa

¶ ¶ ¶

C-terminal deletions

YGGFMRFa YGGFMRF YGGFMRa YGGFMR YGGFMa YGGFM FMRF

0.12

Unrelated peptides

WMDFa pQHPa

0.020 0.20 0.042

Heart 0.90

0.63 1.3 100

10.0 > 1000 > 1000 > 1000 0.034 0.046 0.046 0.070 0.15 0.2• 9.0 10.0 15.0 0.17 1.3 1.3 80 25 > 1000 30

0.05

0.19 8.0 8.5 9.0 0.17

500 > 1000

*Standard one-letter abbreviations for amino acid residues are used, except for the following: nL = nodeucinyl; a= amide; M-OX = oxidized methionyl; ac = acetyl; daY = desaminotyrosinyl; pQ=pyroglutamyl. tBioassays were conducted using Helix hearts as described in Fig. 1. EC~0 values (/zM) were read from log dose-response curves (e.g., Fig. 4); the peptides showed the s a m e Ema x. ¢Competitive displacement experiments were performed with Helix brain or heart membranes as described in the Method section. IC50 values (/xM) were obtained from plots of percent ~2'~I-daYFnLRFamide specifically bound vs. log molar peptide concentration (e.g., Fig. 9). §No effect up to 10 ~M; cardioinhibitory at or above 10/~M. ¶These peptides, at low doses, appeared to be 30-50x more potent than FMRFamide, but were inhibitory above 0.01 to 0.03/zM, (KD=14 nM and Bmax=85 fmol/mg brain) and a low affinity site (Ko=245 nM, Bmax=575 fmol/mg). The specificity of 125I-daYfnLRFamide binding to the high affinity site was e x a m i n e d in competition studies; at the concentration of trace used (0.1 nM), about 72% o f the specific binding consisted of radioligand b o u n d to high affinity sites; the remaining 28% r e p r e s e n t e d binding to the low affinity sites. The high affinity binding was specific for F M R F a m i d e - l i k e pep-

tides; the fragments F M R F and M R F a m i d e were w e a k displacers, and the unrelated peptides met-enkephalin ( Y G G F M ) , gastrin ( W M D F a ) , and thyrotropin-releasing h o r m o n e (pQHPa) did not inhibit binding (Table 1, Fig. 9). Peptides that did c o m p e t e for binding exhibited Hill slopes of about - 0 . 6 (e.g., Fig. 10). This value, along with the biphasic Scatchard analysis (Fig. 8), indicated that the radioligand was not binding to a single class o f independent sites.

1070

PAYZA 6

E

o. "o

o

d Z

50

E

5

Q' "0

0

4

//

,IF-',

30

d Z

3

_z

Z

m

m 0 I,.I. 0 I.M n

40

./

2

20

(,,) I,.i. O 1.1,1 1,1

10

0 0

,

t

20

I

I

40

:

I

60

I

0

I

80

100

120

140

' 1'0' 2'0 ' 3'0' 4'0' 5t0' 6'0' 7t0 '

80

160

[TISSUE],

TIME, minutes FIG. 6. Reversibility of 'zSI-daYFnLRFamide binding to Helix brain membranes. Association: membranes were incubated with 0.1 nM radioligand in the presence or absence of 100/zM acFnLRFamide at 0°C for the times shown, filtered, and the specific binding determined. Dissociation was observed by adding acFnLRFamide (100 /xM final) at the time indicated (arrow), to block radioligand reassociation. Specific binding has been plotted against time for the association (@) and dissociation (&) reactions.

SAR of Helix Brain Receptor Binding The pharmacological specificity of '25I-daYFnLRFamide binding to Helix brain membranes was determined by measuring the potencies (ICs0s) of a series of peptides in displacing the radioligand. The structural changes tested for effects on potency were: amino acid substitutions at all four positions, N-terminal deletions and extensions, and C-terminal deletions. The SAR is summarized in the following list and in Table 1: (1) Substitution of the Phe I residue with tryptophan was completely tolerated (compare W n L R F a to FnLRFa), but rePlacement with tyrosine reduced potency 2.6-fold. Since tyrosine is the least hydrophobic of the three aromatic amino acids, the results suggest that the receptor has a hydrophobic site for binding the Phe 1 residue. (2) Substitution of the methionyl residue had identical effects in both FMRFamide and daYFMRFamide; replacement with norleucine, which closely resembles methionine, produced little change in potency, but the leucine isomer was only about half as effective (Fig. 9). Oxidation of the nonpolar methionine in FMRFamide, which produces a polar sulfoxide or sulfone, reduced potency by nearly 200-fold. (3) Although arginine and lysine both carry a positive charge, the Lys 3 analog had only about 25% the potency of FMRFamide. Furthermore, substitution with the negatively charged aspartate diminished potency by about three orders of magnitude (see tetragastrin-WMDFamide). The results suggest that a positive charge on the R-group is required for

mg

brainlml

FIG. 7. Dependence of lZ'~I-daYFnLRFamidebinding on tissue concentration. Brain membranes (0 to 20 mg initial weight/0.3 ml assay volume) were incubated with 0.5 nM trace in the presence or absense of 100/.tM acFnLRFamide for 2.5 hr at 0°C. Specific binding was determined and has been plotted against tissue concentration.

% 6.

Ox

o

,60

+oo

400

+oo

B, fmol / mg

FIG. 8. Scatchard analysis of ~2'~I-daYFnLRFamidebinding to Helix brain membranes. Membranes were incubated with 0.053 nM 1251daYFnLRFamide in the presence of increasing concentrations of the nonradioactive homolog I-daYFnLRFamide (×) or uniodinated daYFnLRFamide (O), for 2.5 hr at 0°C. Specific binding (B) was determined as described in the Method section, and expressed as fmol peptide bound/mg brain. The B/F ratio (F = molar concentration of free ligand) has been plotted against specific binding. Lines shown were drawn from fits to a two site model [20]: high affinity (Ko=14 nM, Bmax=85 fmol/mg brain); low affinity (KD=245 nM, Bmax=575 fmol/mg brain) (r=0.99).

FMRFAMIDE RECEPTORS IN H E L I X A S P E R S A

1071

I00 -/

m

50

FLRFa

® MRFo

~,

(~) FMRF

o

X

\ X

""q

\

\

X

-,;

X

X

.\

X °

\

"-

~

-6

X

-;

-4

Log Molar Peptide Concentration FIG. 9. Inhibition of lz~I-daYFnLRFamide binding to Helix brain membranes by peptides. Membranes were incubated with 0.1 nM 125I-daYFnLRFamide in the presence of increasing concentrations of peptides. Specific binding was determined and has been plotted against log of the molar peptide concentration. binding at this position, and that a guanidino group is preferred over an amino group. (4) At the Phe 4 position, tryptophan substitution enhanced potency 2.6-fold. In contrast, replacement with a nonaromatic amino acid, leucine, reduced potency 7-fold. As with the Phe ~ position, the region of the receptor that binds this position prefers hydrophobic, aromatic residues. (5) N-terminal deletions virtually obliterated competitive displacement of the radioligand by the resulting fragments (e.g., MRFamide; Fig. 9), indicating a minimal requirement of four residues for binding. (6) In contrast to the deletions, certain N-terminal extensions increased potency in the order: desaminoTyr > Tyr = Tyr-Gly-Gly > acetyl. In addition to the length of an extension, its charge was also important in determining the effect on potency; e.g., addition of tyrosine to the N-terminal of FMRFamide increased potency by 3.5-fold, but removal of the positively charged amino group from the added tyrosine produced the most potent analog of all the peptides tested (daYFMRFamide), which had an ICs0 16-fold better than that of FMRFamide (Fig. 9). Similarly, removal of the positive charge on the amino group of FnLRFamide by N-acetylation increased potency by a factor of three. Other extensions, however, caused an opposite change in potency: the Helix heptapeptides pQDPFLRFamide, NDPFLRFamide, and SDPFLRFamide were only about 5% as active as FMRFamide in displacing the radioligand. This low potency can not be due to overextension at the N-terminal, since the heptapeptide YGGFMRFamide was about equipotent with YFMRFamide. These results suggest that the region of the receptor that binds the N-terminal prefers hydrophobic extensions with missing or blocked amino terminals, but that additions involving Asp-Pro result in a drop in potency, as observed with the Helix heptapeptides. (7) Stepwise C-terminal deletions, beginning with the amide of YGGFMRFamide, resulted in decreases in dis-

1.2-

I,

0.8

0.4

X t

0 i

D 0 ,.J

-0.4 ¸

-0.8 -

-I .2

. -8

.

. -7

. -6

i

-5

-4

Log Molar FMRFomide Concentration FIG. 10. Hill plot for inhibition of 'zSI-daYFnLRFamide binding to Helix brain membranes by FMRFamide. Membranes were incu-

bated with 0.1 nM '25I-daYFnLRFamide in the presence of increasing concentrations of FMRFamide. Specific binding of the radioligand (%B) was determined, and log [%B/(100-%B)] has been plotted against log of the molar FMRFamide concentration. The Hill slope was -0.60. placement potency. At each step the reduction in potency was greater for the unamidated form of the remaining C-terminal residue. The fragment FMRF-OH was nearly 60fold less potent than FMRFamid'e (Fig. 9). The results demonstrate the requirement for the C-terminal amide, and

1072

PAYZA SAR of Helix Heart Receptor Binding

FLRFo

/

FMRFo .TFn L RFo

I m

WnLRf~o" YFMRFoo///7 //YGGFMRFo

-7

///

/// / doYFLRFo .

daYFMI~Fo /

-8

• ocFnLRFo

doYFnLRFQ

DISCUSSION

/ -9

,

-9

The differences between the heart bioassay and brain receptor binding assay, described above in terms of EC.~JICs0 ratios, suggested the occurrence of distinct heart and brain FMRFamide receptor subtypes. That is, that the heart receptors had a relatively greater affinity for the Helix heptapeptides and group I peptides, whereas the brain receptors preferred the group III peptides. To test this hypothesis, membranes from cardiac tissue were used in the binding assay. The potencies of seven peptides in displacing the radioligand from Helix heart membranes were determined. The IC.~0values were nearly identical to those obtained using brain, although FMRFamide itself showed a lower potency for displacing the trace from the heart membranes (Table 1). The overall similarity of binding SAR did not support the notion of distinct heart and brain receptor subtypes, as an explanation for the differences between the heart bioassay and brain receptor binding assay.

-8

- -

-+

- -

Log MolorIC50 FIG. 11. Correlation between inhibition of '~:'l-daYFnLRFamide binding and activity in the isolated heart bioassay. Log molar 1C~,, values (X-axis) were determined in competitive displacement experiments involving Helix brain membranes with various concentrations of FMRFamide analogs. Log molar EC.~(~s(Y-axis) were obtained from log dose response curves for stimulation of the isolated Helix heart. The equation of the solid line (y = 1.64x + 4.10) was obtained by the least squares method (r=0.95). The line of isopotency (dotted line, y = x) has been drawn for comparison.

suggest that the negative charge of an unamidated C-terminal is responsible for the drop in binding potency.

Correlation of Brain Receptor Binding With Heart Bioassay In order to demonstrate the physiological relevance of the binding sites labelled by ~2~I-daYFnLRFamide, the potencies of 10 FMRFamide analogs in the Helix brain receptor binding assay were correlated with their biological activities on the isolated Helix heart (Fig. 11). The line of best fit (solid line, Fig. 11) showed a good correlation (r=0.95) between log [ICs0] and log [ECs0], suggesting that the ~sId a Y F n L R F a m i d e binding sites in the brain membranes were FMRFamide receptors. However, the line of best fit was not parallel to the line of isopotency (broken line, Fig. 11) since the points were clustered above, below, and close to the line of isopotency, constituting three natural groups (Fig. 12). In addition to sharing similar E C J I C s o ratios, the peptides in each group had structural characteristics in common. First, peptides in group I all lacked an N-terminal amino group, and were more potent in the heart bioassay than in the brain receptor binding assay. Thus, the coordinates of these peptides fell below the line of isoptency (i.e., ECs0 < IC50). Group II peptides were substituted or extended with amino acids at the N-terminal, and had similar potencies in both assays. Peptides in Group III had Phe l residues with free amino terminals, and were less potent in the heart bioassay than in the brain binding assay. Thus, these peptides fell above the line of isoptency (i.e., ECs0 > ICs0).

This study demonstrates and characterizes the SAR of FMRFamide receptors in the brain and heart of Helix aspersa. The radioligand ~2'~I-daYFnLRFamide binds reversibly to FMRFamide receptors in brain membranes with a K~) of 14 nM. Scatchard plots of the binding data are curvilinear, and the Hill slopes of peptide competition curves are less than one, suggesting the presence of a second, lower affinity site in the membranes (Kt)=245 riM). The alternative explanation, i.e., the occurrence of different agonist-induced affinity states, is less plausible since negative cooperativity is not observed in log dose-response curves obtained with the isolated heart bioassay. Displacement studies show that the high affinity binding is specific for FMRFamide-like peptides, whereas unrelated peptides do not displace the radioligand. Furthermore, the structure-activity profile of peptides in stimulating the isolated Helix heart is similar to that of displacing '2~I-daYFnLRFamide bound to brain and heart membranes (Fig. 11). The correlation suggests that the high affinity binding sites in Helix brain and heart membranes are FMRFamide receptors. The Helix FMRFamide receptors have SAR characteristics in common with other molluscan bioassays [2, 4-6, 9, 13, 16], i.e., (a) the strong requirement for the C-terminal amide of FMRFamide; (b) the inactivity of N- or C-terminally truncated fragments; (c) the general rank order of effectiveness for amino acid substitutions; and (d) the relative tolerance of substitutions, with amino acids bearing similar R-groups, at the Phe 1and Met" positions compared to the intolerance at the Arg '~. The results suggest that these pharmacological characteristics of molluscan FMRFamide receptors are highly conserved. However, some characteristics of the Helix receptors are distinctive; the most extraordinary feature is the preference of the receptors for FMRFamide derivatives with blocked or extended N-terminals. Of the other molluscan bioassays examined in any detail, only the Macrocallista heart and Geukensia anterior byssus retractor muscle show slight perferences for extended FMRFamide analogs [13]. Further characteristics unique to the Helix receptors are the preferences for methionine over leucine in the Met 2 position, and for tryptophan in both Phe positions. The composition of an N-terminal extension determines the magnitude and direction of its effect on the potency of

FMRFAMIDE RECEPTORS IN H E L I X A S P E R S A

1073 o

I,t_ O:

2.6"

LL 0

It_

2.4-

o

2.2.

rr

J b.

2.0. 2.8. o io (J I-4

o 10 cj bJ

0 tu

rr

1.6. o

1.4-

u_ rr

1.21.0o

o U_ n~

Lu

-J

LL

LL

c-

_j

>-

>-

LL >-

cLL

0 "0

0 "0

0.8-

0 b_

0.6-

j

0.4-

n~

0.20 I

n-

,,iII

E[

FIG. 12. Ratios of peptide potency in the heart bioassay relative to potency in displacing 'z'~I-daYFnLRFamidefrom Helix brain membranes. Ratios of ECJIC~o were calculated, and have been presented as a function of peptide composition. The line of isopotency (dotted line, ECs0/IC.~0=1) has been shown for comparison. peptides binding to the Helix FMRFamide receptors. Certain extensions increase potency; e.g., addition of a daY, YGG, or acetyl group. Enhancement of potency by N-terminal acylation is also a characteristic of neuropeptide Y binding to bovine adrenal medulla membranes, where N-

[propionyl-3H]-NPY binds with about 20-fold greater affinity than NPY itself [8]. The Helix heptapeptides pQDP-, NDP-, and SDPFLRFamide, although they are extended at the N-terminal and bioactive at low concentrations, displace '25I-daYFnLRFamide from Helix brain and heart receptors with a potency about 20 times lower than that of FMRFamide. These results suggest that the Helix heptapeptides cross-react weakly, if at all, at FMRFamide receptors in this snail. The structure-activity profiles of the heart bioassay and the brain receptor binding assay are different in some ways (Fig. 12). The heart receptors may have a slightly lower affinity for FMRFamide than the brain receptors, but the overall similarity in SAR of ~2'~I-daYFnLRFamide binding between brain and heart membranes (Table 1) suggests that the assay differences can not be attributed to putative subtypes of brain and heart receptors. A more likely explanation is that the availability o f a peptide to receptors in the isolated heart may depend on the susceptibility of the peptide to tissue proteolysis, whereas such degradation is inhibited (at 0°C) in the receptor binding assay. This rationale is supported by the observation that the more bioactive peptides (groups ! and II, Fig. 12) are completely or partially protected against aminopeptidase degradation, whereas the biologically less potent peptides (group III) are not; e.g., the relative rate of peptide degradation by aminopeptidase A2 of Mercenaria cardiac membranes is: FMRFa = FLRFa > YGGFMRFa > YFMRFa > > a c F n L R F a (no degradation) [10]. ACKNOWLEDGEMENTS This work was supported by NIH grant HL-28440 and NSF grant DCB-8616356, to M. J. Greenberg. I thank Drs. M. J. Greenberg, S. M. Lambert and D. A. Price for their criticism and suggestions throughout this work. The helpful comments during the preparation of this manuscript, given by Drs. M. J. Greenberg, J. P. Linser and A. H. Neims, are gratefully acknowledged. The assistance of Mrs. Lynn Milstead and Jim Netherton in preparing the figures is greatly appreciated. This is contribution No. 268 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association.

REFERENCES

1. Boyd, P. J. and R. J. Walker. Comparison of the effects of FMRF-amide and pQDPFLRFamide on identified Helix neurons. Cutup Biochem Physiol 86C: 371-373, 1987. 2, Cottrell, G. A., M. J. Greenberg and D. A. Price. Differential effects of the molluscan neuropeptides FMRFamide and the related met-enkephalin derivative YGGFMRFamide on the Helix tentacle retractor muscle. Cutup Biochem Physio175C: 373-375, 1983. 3, Cottrell, G. A. and N. W. Davies. Multiple receptor sites for a molluscan peptide (FMRFamide) and related peptides of Helix. J Physiol 382: 51-68, 1987. 4. Greenberg, M. J. The responsiveness of molluscan muscles to FMRFamide, its analogs and other nueropeptides. In: Molluscan Neuroendocrinology, edited by J. Lever and H. H. Boer. New York: North Holland Publishing Co., 1983, pp. 190--196. 5. Greenberg, M. J., K. Payza, R. J. Nachman, G. M. Holman and D. A. Price. Relationships between the FMRFamide-related peptides and other peptide families. Peptides 8: Suppl 1, in press, 1987. 6. Greenberg, M. J., S. D. Painter and D. A. Price. The amide of the naturally occurring opiate [Met] enkephalin-Arg6-Phe7 is a potent analog of the molluscan neuropeptide FMRFamide. Neuropeptides 1: 30%317, 1981.

7. Greenwood, F. C. and W. Hunter. The preparation of ~3~llabelled human growth hormone of high specific activity. Biochem J 89: 114-123, 1963. 8. Higuchi, H., E. Costa and H.-Y. T. Yang. Neuropeptide Y inhibits the nicotine mediated release of catecholamines from bovine adrenal chromaffin cells. Fed Proc 46: 1448, 1987. 9. Kobayashi, M. and Y. Muneoka. Structural requirements for FMRFamide-like activity on the heart of the prosobranch Rapana thomasiana. Comp Biochem Physiol 84C: 34%352, 1986. 10. Lambert, S. M. and M. J. Greenberg. Proteolysis of FMRFamide: a model of neuropeptide degradation. Soc Neurosci Abstr 12: 1045, 1986. 11. Lehman, H. K. and M. J. Greenberg. The actions of FMRFamide-like peptides on visceral and somatic muscles of the snail, Helix aspersa. J Exp Biol 131: 55-68, 1987. 12. Leung, M. K. and G. B. Stefano. Isolation and identification of enkephalins in pedal ganglia of Mytilus edulis (Mollusca). Proc Natl Acad Sci USA 81: 955--958, 1984. 13. Painter, S. D., J. S. Morley and D. A, Price. Structure-activity relations of the molluscan neuropeptide FMRFamide on some molluscan muscles. Life Sci 31: 2471-2478, 1982.

1074 14. Payza, K. and M. J. Greenberg. Specific binding of ~z~IdesaminoTyr-Phe-norLeu-Arg-Phe-amide to FMRFamide receptors in the brain and heart of Helix aspersa. Soc Neurosci Abstr 12: 827, 1986. 15. Payza, K. and M. J. Greenberg. The structure-activity relations (SAR) of FMRFamide analogs in a receptor binding assay and on cardioactivity in a snail (Helix aspersa) are comparable. Fed Proc 46: 970, 1987. 16. Price, D. A. FMRFamide: Assays and artifacts. In: Molluscan Neuroendocrinology, edited by J. Lever and H. H. Boer. New York: North Holland Publishing Co., 1983, pp. 184-190. 17. Price, D. A., G. A. Cottrell, K. E. Doble, M. J. Greenberg, W. Jorenby, H. K. Lehman and J. P. Riehm. A novel FMRFamide-related peptide in Helix: pQDPFLRFamide. Biol Bull 169: 256-266, 1985.

PAYZA 18. Price, D. A., N. W. Davies, K. E. Doble and M. J. Greenberg. The variety and distribution of the FMRFamide-related peptides in molluscs. Zool Sci 4: 395-410, 1987 19. Price, D. A., K. E. Doble, T. D. Lee and M. J. Greenberg. The distribution of FMRFamide-related peptides in the gastropods. Soc Neurosci Abstr 13: 1076, 1987. 20. Scatchard, G. The attractions of proteins for small molecules and ions. Ann N Y Acad Sci 51: 660-672, 1949. 21. Statistical Consultants, Inc. PCNONLIN and NONLIN84: Statistical analysis of nonlinear models. Am Stat 40: 52, 1986. 22. Voigt, K.-H. and R. Martin. Neuropeptides with cardioexcitatory and opioid activity in octopus nerves. In: Handbook of Comparative Opioid and Related Neuropeptide Mechanisms, vol 1., edited by G. B. Stefano. Boca Raton: CRC Press, 1986, pp. 127-138.